Synergistic antifungal protein and compositions containing same

ABSTRACT

Novel plant proteins (SAFPs) which synergize the activity of antifungal antibiotics are identified. SAFPs are demonstrated to synergize antifungal antibiotics, such as nikkomycins, polyoxins and amphotericins. SAFPs alone also display antifungal activity against several species of fungi, including strains of Candida, Trichoderma, Neurospora and strains of the plant pathogens Fusarium, Rhizoctonia and Chaetomium. Synergistic antifungal compositions containing SAFP and antifungal antibiotics are provided. In particular, synergistic compositions of corn-SAFP (zeamatin), sorghum-SAFP (sormatin) or oat-SAFP (avematin) and nikkomycin are found to be effective as antifungal compositions, especially against the opportunistic human pathogen Candida albicans. Method for employing SAFPs and synergistic compositions containing them for the inhibition of fungi are provided. In addition, a method for purifying SAFP from grain meal is provided.

This invention was made with partial government support under contractnumber DCB 8500233 awarded by the National Science Foundation. Thegovernment has certain rights in this invention.

This is a divisional of application Ser. No. 08/178,708, filed Jan. 10,1994, now U.S. Pat. No. 5,521,153, which is a continuation-in-part ofSer. No. 07/505,781, filed Apr. 6, 1990, now abandoned, which is acontinuation-in-part of Ser. No. 07/104,755, filed Oct. 2, 1987, nowabandoned.

FIELD OF THE INVENTION

This invention relates to novel antifungal plant proteins whichsynergize and enhance the activity of antifungal antibiotics which aredesignated synergistic antifungal proteins (SAFPs), particularly thoseisolated from grains, and especially that isolated from corn (zeamatin).SAFPs alone are useful for the inhibition of growth of certain fungi.The synergistic antifungal compositions of the present invention aregenerally useful in vitro and in vivo for inhibition of fungal growthand for combatting fungal infections. Zeamatin/nikkomycin compositionsare particularly useful in inhibiting the growth of the opportunitieshuman pathogen Candida albicans and for combatting candidal infections.Furthermore, the invention relates to genes for novel antifungalproteins and their use in transgenic technologies.

BACKGROUND OF THE INVENTION

There is significant need for effective antimycotic drugs especially forthe treatment of systemic fungal infections which are life-threatening,common complications in immune-compromised patients, see for exampleHart et al. (1969) J. Infect. Dis. 120:169-191. Among the most virulentorganisms are strains of the yeast Candida, most particularly strains ofC. albicans. While there are several effective topical agents fortreatment of candidiases, treatment of systemic infection is much moredifficult. The drug of choice for systemic infection is amphotericin B,however this drug is highly toxic to the host (see, for example, Medoffand Kobayashi (1980) New Eng. J. Med. 302:145-55). Antimycotic agentsthat are more effective and/or less toxic than existing drugs are highlydesirable.

Several classes of nucleoside antibiotics, including polyoxins (Hori etal. (1971) Agr. Biol. Chem. 35:1280; Hori et al. (1974) Agr. Biol. Chem.38:699; Sasaki et al. (1968) Ann. Phytopathol. Soc. Japan 34:272) andnikkomycins (Dahn et al. U.S. Pat. No. 4,046,881 and 4,158,608; Zahneret al. U.S. Pat. No. 4,287,186; Hagenmaier et al. U.S. Pat. No.4,315,922) have been reported. Polyoxins and nikkomycins are reported tobe useful in agriculture against phytopathogenic fungi and insect pests.Early reports indicated that polyoxins were not effective againstzoopathogenic fungi, such as C. albicans (see, for example, Gooday(1977) J. Gen. Microbiol. 99:1; Shenbagamurthi et. al. (1983) J. Med.Chem. 26:1518-1522). It was believed that the polyoxins were not takenup by target cells. More recently, polyoxins have been reported toinhibit the growth in vitro of certain zoopathogenic fungi including C.albicans and Cryptococcus neoformans when provided at millimolarconcentrations (Becker et al. Antimicro. Agents Chemother. (1983)23:926-929 and Mehta et al. (1984) Antimicro. Agents Chemother.25:373-374). Nikkomycins X and Z have now also been reported to inhibitgrowth of C. albicans in vitro (Yadan et al. (1984) J. Bacteriol.160:884-888; McCarthy et al. (1985) J. Gen. Microbiol. 131:775-780).Polyoxins and nikkomycins are similar in structure and apparently bothact as competitive inhibitors of chitin synthetase (Endo et al. (1970)J. Bacteriol. 104:189-196; Muller et al. (1981) Arch. Microbiol.130:195-197). Chitin is an essential component of the cell wall of mostfungi. Nikkomycins appear, however, to be more effective (about 100fold) against certain fungi, for example C. albicans, than polyoxinswhich is in part due to a higher affinity of nikkomycin for chitinsynthetase and more rapid uptake of nikkomycin by C. albicans cells(McCarthy et al. (1985) supra). The activity of polyoxins andnikkomycins is reported to be inhibited by peptides, such as thosepresent in rich media (Becker et al., 1983, supra; McCarthy et al.,1985, supra; Mehta et al., 1984, supra). Peptides are believed toinhibit uptake of the antibiotic by target cells. The usefulness ofnikkomycins and polyoxins for clinical applications such as in thetreatment of systemic fungal infection, where peptide inhibition islikely, is expected to be limited as the concentrations of antibioticrequired for effective fungal inhibition are not likely to be achievedin vivo.

Mixtures of antimicrobial agents, particularly mixtures in which thecomponents have different modes of action have been used inantimicrobial compositions to broaden activity spectrum or to minimizethe occurrence of resistant strains. Further, certain of these mixturescan display an enhanced antimicrobial activity, greater than theadditive activity of the individual components, due to synergy. Forexample, Gisi et al. (1985) Trans. Br. Mycol. Soc. 85:299-306 reportedthat a number of fungicide mixtures displayed synergistic activityagainst phytopathogenic fungi in field tests. The maximum synergy ratioreported was 7, that is a 7-fold enhancement of activity over thecalculated additive effect. Fungicide mixtures can also show antagonismwith reduced activity of the combination compared to the individualcomponents. It has recently been reported (Hector and Braun (1986)Antimicro. Agent Chemother. 29:389-394) that mixtures of eithernikkomycin Z or nikkomycin X with papulacandin B, an inhibitor ofβ-glucan synthesis, display synergistic antifungal activity againstCandida albicans. Activity enhancements up to about 10 were reported.

Certain enzymes have also been reported to synergize the effect ofantifungal agents. Lysozyme has been reported to synergize the activityof amphotericin B against Candida albicans and Coccidioides immitis(Collins and Pappagianis (1974) Sabouraudia 12:329-340). Naturalmixtures of mycolytic enzymes of fungal origin, designated mycolases,were reported to have a synergistic effect on the activity of theantifungal drugs amphotericin B and nystatin (Davies and Pope (1978)Nature 273:235-6; Pope and Davies (1979) Postgraduate Med. J.55:674-676). The in vitro MICs (minimum inhibitory concentrations) ofthese antifungal drugs were lowered about 5 to 10-fold in combinationswith mycolase. In related in vivo experiments in a mouse model, fungalmycolase was reported to enhance the effectiveness of amphotericin B andnystatin against systemic infection of C. albicans. It was suggestedthat mycolase, which was suggested to be a mixture of carbohydrases,enhanced penetration of the antibiotic into fungal cells. Fungalmycolases, alone, were described as very effective at releasingprotoplasts from Aspergillus fumigatus and C. albicans in vitro and werealso reported to have some effect, alone, against systemic fungalinfection in the mouse model system. In contrast, a prepared mixture ofthe carbohydrases chitinase (β-1,4 N-acetyl-D-glucosaminidase) andlaminarinase (β-1,3(4)-glucanase), while reported to effect protoplastrelease from A. fumigatus and C. albicans, did not enhance theeffectiveness of amphotericin B and nystatin in vivo. Recently, insimilar in vitro and in vivo experiments with fungalmycolase/amphotericin B mixtures, only slight enhancement of antifungalactivity by a fungal mycolase was reported (Chalkley et al. (1985)Sabouraudia 23:147-164). This report suggests that the difference inresults compared to those reported earlier by Davies and Pope (supra)may be associated with the lower chitinase or lower β1,6-D-glucanaseactivities in their preparation of mycolase compared to that employed inthe previous experiments. The specific enzymatic activities present infungal mycolases have not been identified, and the specific protein orproteins in mycolase that may effect antibiotic enhancement have notbeen identified. Some bacterial mycolases have also been reported toeffect enhancements (about 2-fold) of the activity of amphotericin B(Oranusi and Trinci (1985) Microbios 4-3:17-30). Again, no specificenzyme activity was associated with synergy.

Plants appear to have a variety of mechanisms for protecting themselvesagainst infection by viruses, bacteria, fungi and insects. Thesemechanisms are believed to include the presence of inhibitory substancesin plant tissue or plant excretions. Such inhibitory substances may bepresent constitutively in the plant or induced by infection and may below molecular weight compounds such as inhibitins or phytoalexins orcertain proteins, for example, peroxidases, proteinase inhibitors,chitinases or β-1,3-glucanases. In most cases, the inhibitory functionof these substances have not been demonstrated.

In addition to the specific need for more effective, clinically usefulantifungal agents, there is a general need for effective, natural,biodegradable antifungal agents, particularly for use in agricultureagainst plant pathogenic fungi. Such natural antifungal agents may beconsidered to be ecologically preferable to chemical fungicides. To beeconomically useful, especially in agricultural applications, suchnatural antifungal agents should be available in large amounts frominexpensive sources.

It has been recently reported, Roberts and Selitrennikoff (1986)Biochem. Biophys. Acta 880:161-170, that a class of plant proteinscalled ribosome-inactivating proteins (RIPs) are effective againstcertain fungi, for example, Trichoderma reesei. These proteins wereearlier shown to inhibit protein synthesis in animal cell-free extracts(Coleman and Roberts (1982) Biochem. Biophys. Acta 696:239-244). RIPsisolated from grains, including wheat, barley, rye and corn, reportedlyact by enzymatically inactivating the 60S subunit of the animal cellribosome. Coleman and Roberts (1982) reported that RIPs could bepurified to near homogeneity from rye, barley, corn and tritin using thesame procedure (roberts and Stewart (1979) Biochemistry 18:2615-2621).RIPs from rye, barley and tritin were reported to have apparentmolecular weights of approximately 30 kd when analyzed by SDS-PAGE rununder reducing conditions, and corn RIP was reported to run as anapproximately 23 kd protein under similar conditions. Roberts andSelitrennikoff (1986) supra demonstrated that RIP isolated from barleyinactivated Neurospora ribosomes as measured by in vitro inhibition ofpoly(U)-directed polyphenylalanine synthesis in cytoplasmic ribosomepreparations (see also Coleman and Roberts (1981) Biochim. Biophys. Acta659:57-66). These authors have also reported the presence in barley,corn, wheat and rye of another class of antifungal proteins, designatedAFPs which inhibit growth of some fungi, including T. reesei, in vitro.The present work is an extension of this work with plant antifungalproteins.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that a newlyidentified class of plant proteins, designated synergizing antifungalproteins or SAFPs, not only have antifungal activity themselves, butalso significantly enhance the activity of antifungal agents which areinhibitory to the synthesis of fungal cell walls, including polyoxins,nikkomycins and amphotericins. SAFPs can be isolated from grains orgerminating grains, including wheat, rye, barley, sorghum, oats andcorn. SAFP activity has also been detected in other plant seeds, such assoybean. The level of SAFP is enhanced in germinating grains,particularly wheat and rye. Corn is particularly preferred as a sourceof SAFP because active corn-SAFP, herein designated zeamatin, is readilyisolated in partially purified or substantially pure form and inrelatively large amounts. Corn steepwater, for example, was found to bea source of active zeamatin. Furthermore, corn protein extractscontaining corn-SAFP activity were found to be more stable than similarpreparations of rye, wheat or barley, which facilitated isolation andpurification of SAFP from corn. Also preferred is sorghum-SAFP, hereindesignated sormatin. This antifungal protein, like zeamatin, retainsSAFP activity following extraction and isolation in partially purifiedor substantially pure form. Also preferred is partially purifiedoat-SAFP, herein designated avematin. This antifungal protein alsoretains SAFP activity following extraction and isolation in partiallypurified form.

SAFP is itself inhibitory to fungi (including strains of Neurospora,Trichoderma, Phycomyces and Alternaria sp.), yeasts (including strainsof Candida and Rhodotorula), and plant pathogenic fungi (includingstrains of Rhizoctonia, Chaetomium and Fusarium), and particularlyNeurospora crassa and Trichoderma reesei. SAFP is in particularinhibitory to the growth of N. crassa, T. reesei and C. albicans, aswell as to Fusarium monoliforme, F. proliferatum and F. sacchari.

Antifungal activity of SAFP against Neurospora, Trichoderma,Rhizoctonia, Fusarium and Chaetomium has been assessed in in vitro agarplate assays. In similar plate assays, SAFP showed no growth inhibitionof yeasts, including strains of Candida, Rhodotorula and Saccharomyces.However, inhibition assays performed in liquid medium demonstrated thatSAFP itself also inhibited the growth of yeasts, particularly C.albicans. The synergizing activity of SAFP has been assessed in in vitroassays, for example, as the enhancement of the antifungal activity ofnikkomycin Z or nikkomycin X against the opportunistic human pathogen C.albicans.

Zeamatin is an approximately 22 kd protein, as assessed in SDS-PAGEelectrophoresis under reducing conditions. Under nonreducing conditions,zeamatin migrates at 19 kd. The difference in migration rates isbelieved to result from the reduction, under reducing conditions, ofzeamatin's disulfide bonds to produce a less compact conformation.Zeamatin, in substantially pure form or in partially pure form,displays, in particular, synergistic anti-Candida activity andanti-Neurospora activity; and displays antifungal activity againststrains of Trichoderma, Candida, Fusarium, Rhizoctonia and Chaetomium.In particular, corn steepwater and protein concentrates thereof, whichcontain active zeamatin as evidenced by the distinct protein band at 19kd (under nonreducing conditions) and by the presence of synergisticanti-Candida activity, are also found to be inhibitory to the growth offungi, including Trichoderma, Candida, Neurospora and the plantpathogens Fusarium, Rhizoctonia and Chaetomium.

Zeamatin has been isolated in substantially pure form by methodsdescribed herein, as demonstrated by the absence of contaminatingprotein bands in conventional protein gel electrophoresis, as shown inFIG. 4. The N-terminal amino acid sequence (30 amino acids) of zeamatinis provided in Table 5(SEQ ID NO:1). Substantially pure zeamatindisplays no detectable chitinase activity, 1-3 β-glucanase, protease,ribonuclease, phospholipase C, mannanase, N-β-acetylhexosaminidase orribosome-inactivating protein activity as assessed by proceduresdescribed herein or well known in the art. Substantially pure zeamatinpreparations include those in which the 22 kd protein represents about90% or more of the total protein present in the preparation. In in vitrosynergy plate assays, zeamatin was found to greatly enhance theanti-Candida activity of nikkomycin X or Z up to about 100 fold, whilein liquid culture assays, enhancements of up to 1000 fold were observed.Greater enhancement of anti-Candida activity of nikkomycin X or Z isobserved in agar-free medium containing low concentrations of peptoneand peptides. Zeamatin also displayed significant enhancement (about10-fold) of the activity of polyoxin against C. albicans and alsoenhanced (about 3-fold) the activity of amphotericin B against thisyeast.

Sormatin has been isolated in substantially pure form by methodsdescribed herein. Sormatin is an approximately 25 kd protein, asassessed in SDS polyacrylamide gel electrophoresis under reducingconditions. The N-terminal partial amino acid sequence of sormatin isgiven in Table 5(SEQ ID NO:2). Sormatin in substantially pure orpartially pure form, displays synergistic anti-Candida activity and alsodisplays antifungal activity against certain strains. Substantially puresormatin preparations include those in which the 25 kd proteinrepresents about 70-80% or more of the total protein present in thepreparation. Avematin has been isolated in partially purified form bymethods described herein. Avematin is an approximately 22 kd protein, asassessed in SDS polyacrylamide gel electrophoresis under reducingconditions. Avematin displays synergistic anti-Candida activity anddisplays antifungal activity against certain strains.

The present invention discloses a novel class of plant proteins, SAFPs,found in grains such as corn, wheat, barley, sorghum, oats and rye,which, in addition to having antifungal activity, enhance the antifungalactivity of antimycotics, particularly those which inhibit the synthesisof fungal cell walls. In a specific embodiment, the present inventionprovides SAFP isolated from corn, zeamatin, in substantially pure form,having synergistic antifungal activity and antifungal activity. Theinvention also provides partially purified zeamatin preparations havingboth synergistic antifungal activity and antifungal activity. In anotherspecific embodiment, SAFP isolated from sorghum, sormatin, is providedin substantially pure form, having synergistic antifungal activity andantifungal activity. Also provided are partially purified sormatinpreparations having both synergistic antifungal activity and antifungalactivity. In another specific embodiment, SAFP isolated from oats,avematin, is provided in partially purified form, having synergisticantifungal activity and antifungal activity.

SAFP can be employed as an antifungal agent against strains of fungiincluding, among others, Neurospora, Trichoderma, Candida, Fusarium,Rhizoctonia and Chaetomium. Fungal growth inhibition can be accomplishedby applying SAFP, in substantially pure form, in partially pure form, orin crude extracts, to a fungal habitat. The amount of SAFP that isapplied is such that its concentration in the fungal habitat iseffective for growth inhibition of that fungus. The amount of a SAFPrequired for fungal growth inhibition depends on the desiredapplication. The amount of SAFP required for use against a particularfungus in a particular habitat can be readily determined employingappropriate in vitro or in vivo assays that are well known in the art,such as those described herein. For example, it was found that theminimum amount of substantially pure zeamatin required for inhibition ofNeurospora crassa in in vitro hyphal extension inhibition assays wasabout 0.3 g protein/disc. In a similar assay it was found that theminimum amount of substantially pure zeamatin required to inhibitTrichoderma reesei was about 3 g protein/disc. In liquid medium, it wasfound that between about 10 to 30 g/ml of substantially pure zeamatinwas required to inhibit Candida albicans. SAFP can in general beemployed in any fungal habitat in which is retains antifungal activity.Sormatin, avematin and other grain SAFP's are effective for fungalgrowth inhibition at levels comparable to those of zeamatin demonstratedto be effective.

The present invention further provides antifungal compositions whichcontain SAFP in combination with an antifungal antibiotic. SAFP beingpresent in such compositions at a level sufficient to synergize orenhance the antifungal effect of the antibiotic. The antibiotic beingpresent at a sufficient level that the composition has antifungalactivity, i.e. inhibits fungal growth. Synergistic compositions arethose in which the MIC of the antibiotic in the composition is lowerthan the MIC of the antibiotic in the absence of SAFP. The antifungalcompositions of the present invention preferably contain zeamatin,sormatin or oat-SAFP. More preferably are those containing zeamatin andsormatin, and most preferably zeamatin. While, in principle, anyantifungal agent particularly those that inhibit fungal wall synthesissuch as amphotericin B, polyoxin and nikkomycin are useful in thecompositions of the present invention, compositions containingnikkomycins are preferred. Compositions containing nikkomycin X ornikkomycin Z are more preferred and compositions containing nikkomycin Xor nikkomycin Z in combination with zeamatin or sormatin are mostpreferred.

The amount of an SAFP and a particular antifungal antibiotic that incombination produce a synergistic antifungal composition will varydependent upon the desired application of the composition. The amountsof SAFP and antibiotic required in a particular application against aparticular fungus can be readily determined employing appropriate invitro or in vivo assays that are well known to the art such as thosedescribed herein. For example, it was found that compositions containingabout 50 g/ml partially purified zeamatin (fraction CMS) and about 0.06g/ml nikkomycin displayed synergistic antifungal activity, particularlyagainst C. albicans in plate diffusion disk assays (Table 4). It wasfound that concentrations of partially purified zeamatin (fraction CMS)of at least about 10 g/ml in combination with about 0.8 g/ml nikkomycinretained antifungal activity against Candida albicans as measured inplate diffusion disc assays. It was found that concentrations ofsubstantially pure zeamatin of about 0.3 g protein/disc or greater, incombination with concentrations of nikkomycin of about 0.2 g/ml orgreater inhibited growth of C. albicans in diffusion disc assays. It wasfurther demonstrated that concentrations of substantially pure zeamatinfractions of about 0.3 g protein/ml or greater, in combination withconcentrations of nikkomycin of about 0.17 g/ml or greater, inhibitedgrowth of C. albicans in liquid medium.

The synergistic compositions of the present invention are useful ingeneral, as antifungal agents effective against a variety of fungiincluding both phytopathogenic and zoopathogenic fungi. Thesesynergistic compositions are particularly useful against strains ofCandida and Rhodotorula and are most particularly useful against theopportunistic human pathogen Candida albicans. The compositions can ingeneral be employed in any fungal habitat in which the SAFP and theantibiotic retain activity.

The present invention further provides DNA sequences which encode SAFPs.These sequences are useful for expression in transgenic organisms. Inparticular, the invention provides transgenic plants which express SAFPsfrom DNA sequences inserted into plant cells, said sequences being underthe regulatory control of promoters which function in plants. Preferredpromoters are those which are expressed constitutively, in acell-specific or tissue-specific manner, or in response to pathogenattack or wounding. Any promoter expressible in plant cells is, however,a suitable promoter for use in transgenic plants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an elution profile from the initial CM-Sephadex (Trademark,Pharmacia, Piscataway, N.Y.) column purification step of zeamatin fromcorn protein extracts. Protein in each 6 ml fraction was quantified bymeasurement of absorbance at 280 nm. Bound protein was eluted with alinear salt gradient (0.01-0.2M NaCl). One minor and two major proteinpeaks were eluted. The results of anti-Candida albicans synergy assaysof nikkomycin Z by individual fractions are given beneath the proteinfraction profile. Synergy is quantified as strong inhibition (++), weakinhibition (+) and no inhibition (-) of C. albicans in synergy plateassays. Nikkomycin Z synergy was assayed on C. albicans suspensionplates (carrot juice agar medium) by adding 30 l of a 1:10 dilution ofeach column fraction with 25 ng of antibiotic to assay discs. Only thethird peak contained synergistic activity. Fractions 48-55 contained themajority of the desired activity and were combined for furtherpurification.

FIG. 2 shows elution profiles from CM-Sephadex™ column purification ofzeamatin from corn protein extracts. A flow rate of 1 ml/min wasemployed in these separations. Protein in each 6 ml fraction wasquantified by measurement of absorbance at 280 nm. Bound protein waseluted with a linear salt gradient (0.01 0.2M NaCl). Four peaks wereeluted. FIG. 2A displays the quantitative results of antifungal assays,while FIG. 2B displays the quantitative results of enzyme assays acrossthe four peaks. Absorbance at 280 nm is represented in both A and B byclosed circles, solid lines. The results of hyphal extension inhibitionof T. reesei (open circles, solid line), hyphal extension inhibition ofN. crassa (closed squares, dashed lines) and synergistic anti-Candidaactivity (closed triangles, dotted line) are presented on panel A. Theresults of chitinase (closed triangles, dotted fine), glucanase (opensquares, solid line) and β-N-acetyl-hexosaminidase (closed circles,dashed line) assays are presented in panel B.

FIG. 3 is an elution profile of phenyl-Sepharose (Trademark, Pharmacia,Piscataway, N.Y.) column chromatograph purification of zeamatin.Equivalent samples of protein from peak 3 (FIG. 2) were washed throughthe column in 1M ammonium sulfate (open circles) and 0.1M sodiumchloride (closed circles) and bound protein was subsequently eluted with50% ethyl glycol. Protein was quantified by measurement of absorbance at280 nm.

FIG. 4 is a photograph of an SDS-polyacrylamide gel electrophoresis rununder nonreducing conditions of protein fractions from the CM-Sephadex™separation (FIG. 2) and phenyl-Sepharose™ separation (FIG. 3). Panel A,lanes 1 to 5, contain approximately 5 g samples of CM-Sephadex™ columnfractions 28, 35, 43, 48 and 52, respectively. Panel A, lanes 6-8,contain approximately 5 g samples of combined fractions 42-48 from theCM-Sephadex™ column, and phenyl Sepharose™ peaks 1 and peak 2 (FIG. 3),respectively. Five-fold higher concentrations of these same samples arecontained in Panel A, lanes 9-11. Panel B contains two separate phenylSepharose™ column isolates of peak 1 (lanes 1 and 2), two isolates ofpeak 2 (lanes 3 and 4) and a single isolate of peak 3 (lane 5). Panels Aand B also contain molecular weight standards as indicated.

FIG. 5 is an elution profile from the initial CM-Sephadex™ columnpurification step of SAFP from sorghum protein extracts. Protein in eachfraction was quantified by measurement of absorbance at 280 nm. Boundprotein was eluted with a linear salt gradient (0.01-0.4 m NaCl). Fourpeaks were eluted. Nikkomycin Z synergy was assayed on C. albicanssuspension plates (carrot juice agar medium) by adding 30 l of a 1:10dilution of each column fraction with 25 mg of antibiotic to assaydiscs. Only the third peak contained synergistic activity. Peak 3 wasconcentrated using Amicon YM-10 filter.

FIG. 6 is an elution profile of phenyl-Sepharose™ column chromatographpurification of sormatin. Peak 3 (FIG. 5) was loaded onto apre-equilibrated column comprising 1M NaCl, 0.005M sodium phosphate,0.001M EDTA, pH 7.4. The column was washed with 10 ml of the abovebuffer, then eluted with a linear salt gradient (0.01-0.4M NaCl).Protein was quantified by measurement of absorbance at 280 nm. Only peakB contained synergistic activity.

FIG. 7 is a photograph of an SDS-polyacrylamide gel electrophoresis rununder reducing conditions of various protein fractions from theCM-Sephadex™ separation (FIG. 5) and phenyl-Sepharose™ separation (FIG.6) or sorghum extract Lanes 1-5 contain molecular weight standards asindicated, crude extract, 0-35%, 35-65%, and 65-100% ammonium sulfatecuts, respectively. Lanes 6 and 7 contain Peaks 2 and 3 (FIG. 5) fromthe CM-gephadex™ separation, respectively. Lanes 8 and 9 contain Peaks Aand B (FIG. 6) from the phenyl-Sepharose™ separation, respectively.

FIG. 8 includes graphs showing the results of experiments testing theeffect of zeamatin on membrane permeabilization. View (a) is a graphshowing the release of ultraviolet-absorbing material measured asabsorbance at 260 nm from C. albicans as a function of added protein(g/ml). C. albicans was incubated at 37° C. for 30 min with theindicated concentrations of zeamatin (closed circles), lysozyme (closedtriangles), cytochrome C (open squares) or bovine serum albumin (opencircles). View (b) is a graph of the release of radiolabelledaminoisobutyric acid with time from N. crassa on incubation with thegiven agent (12 g/ml) at the indicated temperature (30° C. or 4° C.):zeamatin at 30° C. (closed circles), zeamatin at 4° C. (open circles),amphotericin B at 30° C. (closed squares), amphotericin B at 4° C. (opensquares), pancreatic ribonuclease at 30° C. (closed triangles) andlysozyme at 30° C. (crosses).

FIG. 9 is the CM-Sephadex elution profile of avematin activity. Elutionis performed as described in Example 7. Three peaks were eluted in theseparation. Only peak I was found to contain synergistic antifungalactivity.

FIG. 10 is a photograph of an SDS-gel electrophoresis run under reducingconditions containing the CM-Sephadex peak I from the oat-SAFPpurification in FIG. 9. Lane 1 contains molecular weight standards asindicated. Lane 2 is the avematin CM-Sephadex peak I and Lane 3 containspeak 3 of the CM-Sephadex separation of corn extract containingzeamatin.

DETAILED DESCRIPTION OF THE INVENTION

The term synergy as used herein applies to the enhancement of antifungalactivity of certain antibiotics by certain plant proteins, SAFPs.Synergy can be quantitatively measured as the lowering of the minimuminhibitory concentration (MIC) of an antibiotic effected by combining itwith an SAFP and is specifically measured herein as enhancement of theactivity of antimycotics against Candida albicans in in vitro assays.When used herein, the term significant enhancement of antifungalactivity refers to enhancements of 10-fold or greater. The MIC isgenerally defined as the highest dilution (i.e. lowest concentration) ofan agent that inhibits growth of a microorganism. In liquid medium, theMIC is usually defined as the lowest concentration of an agent whichprevents visible growth of a standard inoculum which is measure byculture turbidity. Inhibition plate assays in which discs impregnatedwith an antimicrobial agent are placed on microbial lawns can also beused to access MICs (diffusion disc assays). As described in Example 2,the MIC in disc diffusion assays is defined as the lowest concentrationof an agent applied to a disc which gives a measurable zone of growthinhibition of the microbial lawn. MICs are determined empirically andoften display strain and media dependence.

The effectiveness of an antibiotic agent in vivo is generally assessedin animal model systems, such as those described in Pope and Davies(1979) supra; and Chalkley et al. (1985) supra. In such experiments,effectiveness is assessed as survival or cure rate. Comparisons of theeffectiveness of different antibiotic agents is assessed as increases insurvival or cure rates.

The present work is an extension of experiments with antifungal proteins(AFPs) which were isolated from barley, corn and wheat (Roberts andSelitrennikoff (1988) J. Gen. Microbiol. 134:169-176). These proteinsinhibited growth of Trichoderma, Phycomyces and Alternaria and have beenshown to have endochitinase activity. Wheat and barley AFP chitinasesdid not inhibit growth of Neurospora, in contrast to corn AFPpreparations. Growth of the important human pathogen Candida albicanswas found to be resistant to inhibition by the AFPs in agar plateassays. AFPs were then assessed to determine if they synergized withantifungal antibiotics to lower the MICs of the antibiotics. Selectedresults of such experiments are summarized in Table 1. Nikkomycin, amixture of nikkomycin Z and X, synergized with all AFP preparations, butsynergy was particularly dramatic with corn-AFP preparations. Polyoxinsynergized significantly with corn and wheat AFP preparations, whilemodest synergy was observed with combinations of amphotericin and AFPpreparations from barley and corn. In contrast, no synergy was observedwith papulocandin and AFP preparations. Wheat and barley AFPs (Table 1)were purified to homogeneity. The corn-AFP preparation (Table 1) whenchromatographed through a CM-Sephadex™ column was shown to containmultiple protein peaks (FIG. 1). Using synergy with nikkomycin toinhibit the growth of C. albicans as an activity assay, the synergizingactivity in corn-AFP preparations was found to reside in a singleprotein fraction from CM-Sephadex™ column chromatography, see FIG. 1.Further purification of this fraction using conventional hydrophobiccolumn chromatography with phenyl-Sepharose™ resulted in the isolationof an approximately 22 kd protein. The 22 kd protein which effectedstrong enhancement of nikkomycin activity was designated a corn-SAFP,and specifically named zeamatin.

                  TABLE 1                                                         ______________________________________                                        Effect of AFP preparations on Antibiotic MIC against Candida albicans         MIC against Candida albicans (g/disc)                                         AFP.sup.a                                                                            Nikkomycin                                                                              Papulacandin                                                                            Polyoxin B                                                                            Amphotenicin B                             ______________________________________                                        None   0.17      0.5       50.0    5.0                                        Barley 0.05      0.5       50.0    1.6                                               (3×).sup.b            (3×)                                 Blue corn                                                                            <0.0017   0.5       5.0     1.6                                               (>100×)       (10×)                                                                           (3×)                                 Yellow <0.0017   0.5       5.0     1.6                                        corn   (>100×)       (10×)                                                                           (3×)                                 Wheat  0.05      0.5       5.0     5.0                                               (3×)          (10×)                                        ______________________________________                                         .sup.a AFP preparation was supplied at 15 g protein/disc. AFP fractions       were prepared as described in Roberts and Selitrennikoff (1987) J. Gen.       Microbiol., supr.                                                             .sup.b The number in parentheses refers to the fold reduction in MIC     

Since a significant loss in specific synergizing activity was observedin the conventional phenyl-Sepharose™ chromatography step, efforts weremade to improve the purification of corn-SAFP activity. Improvedpurification of zeamatin was obtained by carrying out the CM-Sephadex™chromatography at a slower flow rate than had been employed in previousseparations and more importantly, by employing a novel phenyl-Sepharose™chromatographic procedure. Slower elution in the CM-Sephadex™ stepresulted in four distinct protein peaks (FIG. 2) rather than the threepeaks observed previously (FIG. 1). Synergistic anti-Candida activitywas found only in peak 3. Anti-Neurospora activity was also confined topeak 3, while anti-Trichoderma activity was observed in all peakfractions. All four peaks were also assayed for chitinase, glucanase andβ-N-acetylhexosaminidase activity. None of these enzyme activitiescoincided with the anti-Neurospora or synergistic anti-Candida activityof peak 3.

Zeamatin was then further purified employing a novel method ofhydrophobic column chromatography. Fractions from the CM-Sephadex™column that contained synergistic anti-Candida activity were combinedand subjected to phenyl-Sepharose™ column chromatography. Thisseparation was carried out by loading the column at a lower saltconcentration than is typically employed in order to reduce thehydrophobic interactions between the proteins and the column. Boundprotein was then eluted with 50% ethylene glycol. This procedureresulted in the profile of FIG. 3 containing three bands, one of whichpassed directly through the column (1), a second which was somewhatretarded (2), and a third smaller band which was eluted with 50%ethylene glycol (3). SDS-PAGE electrophoresis (FIG. 4) demonstrated thatpeak 2 from the low salt phenyl-Sepharose™ separation contained anapparently homogeneous 19 kd protein (zeamatin), as determined bygel-electrophoresis under nonreducing conditions. When run ingel-electrophoresis under reducing conditions, this 19 kd protein ran asan apparently homogeneous 22 kd protein. This peak 2 was alsodemonstrated (see Table 2) to contain all synergistic antifungalactivity as well as all anti-Neurospora activity. Peak 2 also containedanti-Trichoderma activity.

                  TABLE 2                                                         ______________________________________                                        Antifungal and Enzymatic Activities                                           of Proteins Purified by Phenyl-Sepharose ™ Chromatography                                           Anti-Tri-                                                                            Anti-   Anti-                                 Fraction                                                                             Chitinase                                                                              Glucanase                                                                              choderma                                                                             Neurospora                                                                            Candida                               Assayed                                                                              Activity.sup.a                                                                         Activity.sup.b                                                                         Activity.sup.c                                                                       Activity.sup.c                                                                        Activity.sup.c                        ______________________________________                                        Combined                                                                             3.5      84       0.5    1       0.9                                   fractions                                                                     (FIG. 3)                                                                      Peak 1.sup.d                                                                         2.9       9       0.5    >5      >10                                   Peak 2.sup.d                                                                         NA.sup.e  1       3      0.3     0.2                                   Peak 3.sup.d                                                                         NA.sup.e 2010     NA.sup.e                                                                             NA.sup.e                                                                              --                                    ______________________________________                                         .sup.a Reducing sugar released after incubation at 37° for 4 h         (moles glucose/mg protein).                                                   .sup.b Reducing sugar released after incubation at 37° for 20 min.     (moles glucose/mg protein).                                                   .sup.c Minimum amount of protein required to inhibit fungal growth (g         protein/disc) in the presence of subinhibitory levels of nikkomycin.          .sup.d Protein peaks from chromatography in 0.1M NaCl (FIG. 2)                .sup.e NA = No activity detected.                                        

The association of synergistic anti-Candida activity with zeamatin wasconfirmed by a bioautography experiment in which substantially pureprotein of peak 2 (FIG. 3) was subjected to electrophoresis of pH 6.0 ina nonreducing acrylamide gel. The protein from this gel was allowed todiffuse into an agar plate containing freshly seeded C. albicans andsub-inhibitory concentrations of nikkomycin. A strong zone of growthinhibition was observed only at a position in the agar which coincidedwith the zeamatin protein band. This bioautography procedure has alsobeen employed to demonstrate the association of synergistic anti-Candidaactivity with the sormatin and avematin protein bands.

Early experiments demonstrated that zeamatin could be extracted fromcornmeal using 0.05M acetic acid, and that it was stable at moderatetemperatures. Thus, it seemed possible that corn-SAFP might survive thelow pH and elevated temperatures of the corn steeping process and bepresent in an active form in steepwater. Accordingly, steepwater sampleswere obtained from the Adolph Coors corn refining plant, Johnstown,Colo., and analyzed for antifungal activity. The 35-hour lightsteepwater sample was effective at inhibiting growth of Trichodermareesei and Candida albicans in the presence of sub-inhibitory levels ofnikkomycin. This activity was lost following heating at 90° C. for 15minutes. Analysis of the proteins in steepwater by SDS-PAGE undernonreducing conditions showed a broad protein smear accompanied by adistinct protein band at 19 kd. This protein band corresponds with the22 kd zeamatin band observed under reducing conditions. The synergisticanti-Candida activity of the extract indicated that zeamatin was intactand active in steepwater. These experiments are of interest because theyidentify corn steepwater as a potential commercial source for the largescale isolation of zeamatin.

Additional experiments showed that the synergistic antifungal andantifungal activity in steepwater could be precipitated using ammoniumsulfate, ethanol, or acetone. Moreover, testing these concentratedpreparations on a number of fungal plant pathogens showed that theyinhibited growth on agar of pathogenic strains of Trichoderma,Rhizoctonia, and Fusarium.

SAFPs have been identified in sources other than corn. Seeds are knownto synthesize large amounts of new enzymes (e.g., glucanases) ongermination. Accordingly, wheat and rye were allowed to germinate forthree days, after which protein extracts were prepared as in AFPpreparations. These protein extracts were found to contain high SAFPactivity and were found to lower the MIC of nikkomycin against C.albicans by about 100-fold. The wheat and rye SAFPs could be partiallypurified by the same procedure used for corn-SAFP. However, the wheatand rye SAFP preparations, in contrast to preparations from corn, lostactivity after several days storage at 4° C. and have not as yet beenfurther characterized.

Similar to zeamatin preparations, sorghum and oat extracts were found tocontain SAFP activity that was stable. Sormatin was purified to apparenthomogeneity using methods similar to those employed to purify zeamatin.Avematin has been partially purified to homogeneity or near homogeneityfrom partially purified materials described herein by application byapplication of methods applied to the purification of zeamatin orsormatin, or by application of methods known to the art of proteinpurification.

Chitinase and glucanase preparations from several other sources werealso tested in the synergy assay. No synergy with nikkomycin was foundwith chitinases from Serratia marcescens, Pseudomonas stuzeri, orStreptomyces griseus or in glucanase preparations from Penicillium ormollusk. Significant synergy was observed, however, with a partiallypurified glucanase preparation from the fungus Rhizopus and incommercial bacterial (Arthrobacter luteus) enzyme mixture containingboth chitinase and glucanase called Zymolase (available from SigmaChemical Co., St. Louis, Mo.). The nature of the synergizing enzymes inthese preparations has not been identified, and it is not known whetherthey act by a mechanism that is similar to plant SAFPs. The synergizingactivity in these preparations may be due to minor components in themixtures.

The anti-Candida synergy that is observed with the SAFP/antibioticcompositions of the present invention is surprising, since it is notpredictable that a particular combination of two antimicrobial agents,even those which have different modes of action, win be synergistic.

The very strong synergy observed in the zeamatin/nikkomycin compositionsof the present invention against Candida strains was also surprising.Typically, enhancements due to synergy an observed to be in the range of10 fold or less. For the zeamatin/nikkomycin compositions, the MIC ofnikkomycin was lower by up to 100 fold in plate assays. In similarinhibition assays of Candida albicans done in liquid media, the MIC ofnikkomycin was also lowered by up to about 100 fold (Table 3).

                  TABLE 3                                                         ______________________________________                                        Growth Inhibition of C. albican in Liquid Culture                             Nikkomycin                                                                             Zeamatin in Wells (g/ml).sup.A                                       in Wells (g/ml)                                                                        0      0.3    1.0  3.0  10   30   100  300                           ______________________________________                                        0        +++    +++    +++  +++  +    -    -    -                             0.017    +++    +++    +++  +++  -    -    -    -                             0.050    +++    ++     ++   ++   -    -    -    -                             0.17     +++    -      -    -    -    -    -    -                             0.5      +++    -      -    -    -    -    -    -                             1.7      +++    -      -    -    -    -    -    -                             5        +      -      -    -    -    -    -    -                             17       -      -      -    -    -    -    -    -                             ______________________________________                                         .sup.A Each well contained 150 l of 2% carrot juice inoculated with C.        albicans suspension to give an absorbance at 630 nm of 0.005. Fungal          growth was scored as +++, ++, + or no growth (-) after visual inspection      for turbidity.                                                           

Zeamatin/nikkomycin compositions were found to be effective againstseveral strains of C. albicans which varied in their sensitivity towardnikkomycin (Table 4). In each case, zeamatin significantly synergizedthe effect of nikkomycin and lowered the MIC of nikkomycin incompositions by about 33 to 100 fold. Zeamatin/nikkomycin compositionswere found to be effective against Candida albicans on either poor orrich medium. Zeamatin synergized nikkomycin activity of nikkomycin hasbeen found to be attenuated by peptide inhibition.

The mechanism by which SAFP synergizes the action of polyoxins,nikkomycins and amphotericins is not known. It was thought that SAFPmight act to increase penetration of the antibiotics into the targetfungi. This could occur as the result of degradation or permeabilizationof the fungal cell wall by SAFP. Fungal cell walls are composed ofchitin, glucans with β-1,3 or β-1,6-linkages and mannans with α-1,6,α-1,2 or α-1,3-linkages. It has been demonstrated, however, thatzeamatin, unlike other antimycotic agents, does not have chitinase,glucanase or mannanase activity. A more probable mechanism, supported byexperiments described below, is that SAFP permeabilizes the fungal cellmembrane. It is suggested that SAFP lyses fungi by direct insertion ofthe protein into fungal membranes to form transmembrane pores.Amphiphilic polypeptides may bind to cells through a cationic region ofthe molecule followed by insertion of a hydrophobic domain through thelipid bilayer of the membrane. For example, zeamatin's amphiphilicnature is suggested by the protein's late elution from CM-Sephadex™ (acationic property) and its retarded passage through phenyl-Sepharose™ (ahydrophobic property). That zeamatin acts via cell membranepermeabilization is further supported by the rapid effect of lowconcentrations of SAFP's on fungi, even at 0° C. For example, 1 g/mlzeamatin induces hyphal rupture in less than 15 seconds at 23° C. Thisrapid rapture suggests a non-enzymatic mechanism of action. Theoperability and utility of the SAFPs of the present invention are,however, not dependent upon these suggested mechanisms, and the practiceof the present invention does not require characterization of thespecific activity of an SAFP. Similarities in structure (similarmolecular weights, similar elution behavior on chromatography andhomologies in N-terminal sequence) of zeamatin, sormatin and avematin,and more importantly, their common function in synergism of anti-fungalactivity, indicate that these proteins and other protein strainsdisplaying this function represent a class of proteins (SAFPs) which actby an analogous mechanism.

In the count of conducting experiments to determine zeamatin's mechanismof action on fungi, the ability of zeamatin to inactivate ribosomes wasassessed. Ribosome inactivating proteins (RIPs) had been isolated fromgrains including corn (Coleman and Roberts (1981) supra). Zeamatin wastested as described in Coleman and Roberts (1981) supra, for its abilityto inhibit protein synthesis in Neurospora cell-free extracts. Additionof up to 10 g/ml of zeamatin to this in vitro protein synthesis assayhad no inhibitory effect. In contrast, RIP isolated from corn (Colemanand Roberts (1981) supra) inhibited ribosomes at a level of 0.06 g/ml.Furthermore, corn-RIP purified as described in Coleman and Roberts(1981) supra was found to have no synergistic antifungal activity inCandida plate assays employing nikkomycin. Specifically, addition of upto 1500 g/ml of corn-RIP displayed no synergy with nikkomycin. It shouldbe noted that greater than 90% of corn-RIP activity was found to residein the 55%-85% ammonium sulfate fraction prepared from cornmeal extract,while 90% of the zeamatin activity was found to reside in the 30%-55%ammonium sulfate fraction.

Since purified zeamatin contained no detectable enzymatic activityassociated with fungal cell wall degradation, the action of zeamatin onthe fungal cell membrane was examined. Several experimental approacheswere used to examine whether the fungal membrane was the site of actionof zeamatin (and by analogy the site of action of other SAFPs fromgrain). The release of ultraviolet-absorbing material from suspension ofC. albicans was examined (see FIG. 8a). It was found that as littlezeamatin as 1 g/ml produced detectable cell leakage. A second leakageexperiment (FIG. 8b) was performed by preloading N. crassa cells with ¹⁴C!-aminoisobutyric acid, a non-metabolizable amino acid used to measuremembrane integrity (Georgopapadakou et al. (1987) Antimicrob. AgentsChemother. 31:46-51) and following its release from cells upon treatmentwith zeamatin or amphotericin B, which antibiotic is known topermeabilize fungal membranes. Incubation of N. crassa with zeamatin at30° C. caused an immediate release of radioactivity which was completeby 3 min. At 0° C., release was slower, but 80% of the radioactivity waslost from cells by 20 min. By comparison, amphotericin B produced fairlyrapid leakage at 30° C., but no cell leakage at 0° C. (It had been notedby Gale (1974) J. Gen. Microbiol. 80:451-465 that amphotericin did notpermeabilize cells at 0° C. and this effect was attributed to immobilityof the membrane lipid and or a requirement for metabolic energy.) Thebasic proteins lysozyme and pancreatic ribonuclease were found to haveno effect on membrane permeability of N. crassa. Zeamatin was found bymicroscopic examination to induce hyphal rupture in germinated spores ofN. crassa. Incubation with zeamatin produced hyphae that stained withmethylene blue and were vacuolate. Hyphal rupture occurred in less than15 s at 23° C. with as little as 1 g/ml zeamatin. Most, but not all,ruptures were observed at hyphal tips or immediately behind the hyphalapical dome, which are regions that are susceptible to turgor pressure.These results indicate that SAFP acts to permeabilize the fungal plasmamembrane.

Certain basic proteins had previously been shown to cause release ofcytoplasmic components from Candida (Yphantis et al. (1967). However, incontrast to the effect of SAFP, the protein concentrations required tocause such release are relatively high as can be seen from the limitedrelease caused by additions of 40 g/ml lysozyme, cytochrome C or bovineserum albumin (FIG. 8a).

The results of the release experiments of FIG. 8 and the effect of SAFPon hyphae are consistent with a fungal lysing mechanism mediated bydirect insertion of the SAFP protein into fungal membranes to formtransmembrane pores. A variety of proteins have been shown to lysemammalian cells by such a mechanism ((Yphantis et al. (1967) J.Bacteriol. 94:1509-1515; Bhakdi and Tranum-Jensen (1987) Reviews ofPhysiology, Biochemistry and Pharmacology 107:147-223). In addition,polypeptides and proteins have been isolated from a number of sourceswhich appear to lyse microorganisms by a similar mechanism, includingmelittin (bee venom, Mackler and Kreil (1977) Inflammation 2:55-65),cecropins (insect haemolymph, Steiner et al. (1985) Nature 292:246-248),magainins (toad skin, Zasloff (1987) Proc. Natl. Acad. Sci. 845449-5453)and thionins (Bohlmann et al. (1988) EMBO J. 7:1559-1565). Thioninsdiffer from AFPs of the present invention In that they are described asrelatively low molecular weight polypeptides (˜5,000 Mv) occurring inseeds and leaf tissue in cereals, such as barley. Further, they aredescribed as having a general toxic effect on bacterial, fungi and smallanimals. Synergistic antifungal activity has not been attributed tothionins. Finally, thionins have no apparent sequence homology to SAFPs.In addition, larger proteins like the bacteriocins: colicin (Parker etal. (1989) Nature 337:93-96) and halocin (Torrebianca et al. (1989) J.Gen. Microbiol. 135:2655-2661) and the complement attack complexdescribed by Bhakdi and Tranum-Jensen (1987) supra are reported to actby this mechanism. Many of these polypeptides and proteins haveamphiphilic properties, and may act by binding to cells through acationic region of the molecule followed by insertion of a hydrophobicdomain through the lipid bilayer of the membrane.

Substantially pure zeamatin and sormatin were subjected to conventionalprotein sequencing techniques. An initial determination of the partialamino acid sequences of these proteins is given in Table 5. A computersearch for protein sequences having homology to the SAFPs was performed.Homology was found to thaumatin, an intensely sweet protein fromThaumatococcus danielli (see, Edens et al. (1982) Gene 18:1-12),pathogenesis-related (PR) proteins, for example from tobacco (see,Cornelissen et al. (1986) Nature 321:531-532) and osmotin, a tobaccoprotein associated with the plant's adaptation to salt (Singh et al.(1987) Plant Physiol. 85:529-536). Particularly striking homology wasfound between the N-terminal region of zeamatin and sormatin and that ofa maize proteinase/amylase inhibitor, designated MAI, reported byRichardson et al. (1987) Nature 327:432-434. A comparison of theN-terminal sequences of MAI, thaumatin, tobacco PR and osmotin isincluded in Table 5. The sequence reported in the Richardson et al.(1987) reference (supra) is attributed to a maize protein which displaysin vitro inhibition of bovine trypsin and insect α-amylases arereportedly not inhibited by MAI.) This maize bifunctional inhibitor,MAI, is reported to contain 206 amino acids which corresponds to a Mr of22,077 and is noted to contain a high content of cystein. The N-terminalamino acids of zeamatin. Sormatin differs from zeamatin and MAI at 2amino acids in the first 22 N-terminal amino acids. The sequencehomologies observed between the SAFPs and MAI is surprising, given theirdistinct biological activities. Substantially pure zeamatin and sormatinhave been assayed for inhibition of trypsin as described in Richardsonet al. (1987) supra, and found to have no such activity. No antifungalor antimicrobial activity has been attributed to MAI. Richardson et al.(supra) pointed out that MAI had significant homology to thaumatin andPRs induced in tobacco plants in response to viral infection. Thefunctions or activities of thaumatin and PRs have not yet beendetermined. Richardson et al. suggested that thaumatin and PRs and otherproteins homologous to MAI should be assessed for inhibition ofhydrolytic enzymes. Richardson et al. note that other workers (Pierpontet al. (1987) Physiol. Mol. Pl. Pathol. 31:(2)291-298 were not able todetect trypsin inhibition by PR.

From the amino acid sequence of zeamatin determined as described above,oligonucleotides were designed for the purposes of cloning the zeamatincDNA using the polymerase chain reaction (PCR) and the screening of acDNA library. The cloned and sequenced zeamatin cDNA is shown in Table 7(SEQ ID NO:7). Using similar techniques of protein sequencedetermination, PCR and library screening cDNAs for other SAFPs includingsormatin and avematin are similarly obtainable. These cDNAs areexpressible in transgenic organisms under the control of promoters whichare known to be active in the target cells. Thus, for expression intransgenic plants the cDNAs can be inserted into plant cells under thecontrol of a promoter known to be expressible in plant cells. Typicallysuch an "expression cassette" would have the following components: (1) apromoter expressible in plant cells, (2) DNA sequence encoding the SAFPprotein, or sequence with substantial homology thereto, and (3) atranscriptional terminator. SAFPs such as zeamatin may already includein their cDNA an aminoterminal sequence which serves to target themature SAFP to the apoplast. However, where such a component of the cDNAmay be absent this may be added to the expression cassette by including:(4) a corresponding extracellular targetting sequence encoding a signalpeptide from a different gene (e.g. PR1) fused to the amino terminus ofthe SAFP. Alternatively, for cytosolic localization this sequence may beomitted or disrupted if a native signal peptide exists. A furthercomponent which can be included in the expression cassette is (5) asequence fused to the carboxyterminus, typically required together withthe amino terminal sequence of (4) which may serve to target the SAFP tothe cell vacuole. Techniques for the transfer of expression cassettes toplant cells are well known in the art. Preferred procedures fordicotyledonous plant species involve the use of Agrobacteriumtumefaciens and corresponding binary vectors (Alexander et al., (1993)PNAS 90:7327-7331). Preferred procedures for monocotyledonous plantspecies involve direct gene transfer with corresponding vectors. See,for example, Koziel et al., Biotechnology 11:194-200 (1993).

Zeamatin and sormatin are not sweet-tasting and no antifungal activitycould be demonstrated with thaumatin against N. crassa, C. albicans orT. reesei. No synergistic antifungal activity against C. albicans couldbe demonstrated with thaumatin.

It is important to note when considering the relationship betweenprotein structure and function that small structural changes instructure are known to affect antifungal activity. For example, thehuman neutrophil defensin HNP-1 effectively kills C. albicans, whereasHNP-3, which differs from HNP-1 only in its amino terminal amino acid,does not (Selsted et al. (1985) J. Clin. Invest. 76:1436-1439).

                  TABLE 4                                                         ______________________________________                                        Comparison of                                                                 Growth Inhibition of Various strains of Candida albicans on                   Different Growth Media                                                               MIC Nikkomycin Z.sup.1                                                        Carrot Juice Agar                                                                           Nutrient Agar                                            Candida         Nikk/             Nikk/                                       strain   Nikk   Zeamatin.sup.2                                                                             Nikk Zeamatin.sup.2                              ______________________________________                                        I.sup.3  170    1.7          1500 15                                          II       170    1.7          5000 50                                          III       50    0.5           500 15                                          IV        17    0.5           170  5                                          ______________________________________                                         .sup.1 Minimum Inhibitory Concentration (MIC) of nikkomycin Z in units of     ng/disc. All plates were incubated for 16 h at 37° C.                  .sup.2 In assays of Nikk/zeamatin, the concentration of zeamatin was 15       g/disk. Zeamatin was partially purified through the CMSephadex.sup.™       step (fraction CMS).                                                          .sup.3 I is laboratory strain B311 and strains II-IV are separate clinica     isolates.                                                                

                                      TABLE 5                                     __________________________________________________________________________    N-Terminal Amino Acid Sequences of Grain SAFPs and Several Homologous         Proteins.sup.A,B                                                              __________________________________________________________________________    Sormatin                                                                           AVFTVVNRCPYTVWAASVPV-----GG       (SEQ ID NO:2)                          Zeamatin                                                                           AVFTVVNQCPFTVWAASVPV-----GGGRQLNRGE                                                                             (SEQ ID NO:1)                          MAI  AVFTVVNQCPFTVWAASVPV-----GGGRQLNRGE                                      TPR  ATFDIVNQCTYTVWAAASP------GGGRQLNSGQ                                      THA  ATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGE                                      Osmotin                                                                            ATIEVRNNCPYTVWAASTPI-----GGGRRLDRGQ                                      __________________________________________________________________________     .sup.A MAI is the bifunctional inhibitor described in Richardson et al.       (1987) supra; TPR is a tobacco pathogenesisrelated protein; and THA is        thaumatin, whose sequence is taken from that given in Richardson et al.       (1987) supra; osmotin sequence is taken from Singh et al. (1987) supra.       .sup.B Sequences are aligned for maximum overlap.                        

The following examples are intended for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1

Isolation and Purification of Zeamatin from Corn

Antifungal protein (AFP) extracts, containing SAFP and other antifungalproteins, were prepared from corn using methods similar to thosedescribed in Roberts and Selitrennidoff, 1986 (supra). The initialprotein extraction was carried out at essentially without loss ofactivity. Corn meal was obtained either from the refrigerator section ofhealth food stores or by grinding dried corn finely in a coffee grinder.AFP could be extracted from corn meal using either acidic or neutral pHbuffers (pH ranging from about 4.0 to 7.0) In a typical extraction, cornmeal (2 lbs) was stirred for 1 h at 40° C. 2 l of 50 mM NaCl/25 mMsodium phosphate (pH 7.0)/5.0 mM EDTA. The resulting suspension wascentrifuged (10,000×g, 20 min, 4° C.) and the supernatant proteinextract was saved.

Ammonium sulfate was slowly added with stirring to the corn proteinextract, to obtain a 30% saturated solution which was left overnight at40° C. The solution was then centrifuged (10,000×g, 20 min) and thesupernatant was made 55% saturated with ammonium sulfate. The solutionwas left 20 min at 4° C. and centrifuged (10,000×g, 20 min). Theprecipitate pellet from the 55% saturated ammonium sulfate solution wassaved and then resuspended in 80 ml of 10 mM NaCl/5.0 mM sodiumphosphate (pH 7.0)/1.0 mM EDTA resuspension buffer. The resuspended30%-55% protein fraction was then clarified by centrifugation anddialyzed (16 h, 4° C.) against 2×1 l of resuspension buffer.

The dialyzed solution was again clarified by centrifugation prior tofurther purification by chromatography on carboxymethyl-Sephadex™(CM-Sephadex™). CM-Sephadex™ column chromatography was carried out at23° C. The dialyzed protein fraction, was passed through a CM-Sephadex™column (C-50-120, from Sigma Chemical Co., St. Louis, Mo) equilibratedwith resuspension buffer. The column was prepared by soaking 2 g ofcolumn material in resuspension buffer and adding the slurry to a columnto form approximately 60 ml of packed gel volume. The dialyzed proteinfraction was washed through the column with buffer until the absorbanceof the effluent at 280 nm had fallen to approximately 0.2; typicallythis required washing with about 400 ml of buffer. In a typicalpreparation, about 1700 mg of protein was added to the column, of which2/3 washed through and 1/3 remained bound. All of the AFP activityremained bound to the column.

Antifungal protein was then eluted from the column using a linear saltgradient prepared conventionally by running 220 ml of 200 mM NaCl/5 mMsodium phosphate (pH 7.0)/1 mM EDTA into 220 ml of resuspension buffer.Fractions (6 ml) were collected at 3-minute intervals. This gradienteluted three protein peaks (as assayed by absorbance at 280 nm) as shownin FIG. 1; one minor component eluting early and two major componentseluting later. The latest eluting peak contained all of the SAFPactivity. Fractions containing the highest synergistic antifungalactivity, fractions 48-55 (FIG. 1), were collected, and concentrated3-fold by ultra-filtration (Amicon YM-10 filter). This fraction,designated fraction CMS, retained antifungal and synergistic antifungalproperties and was employed in the in vitro anti-Candida plate assayspresented in Table 4 (infra).

Fraction CMS retained antifungal activity against T.reesei, but did notinhibit C. albicans in agar plate assays. This fraction was found tocontain all synergistic antifungal activity against C. albicans. Thisfraction as assayed by SDS-PAGE was found to contain several proteinpeaks, including a peak at about 19 kd (by SDS-PAGE under nonreducingconditions).

Further purification of fraction CMS was initially attempted employingphenyl-Sepharose™ column chromatography employing 1M (NH₄)₂ SO₄ at 4° C.Two protein peaks (assayed by absorbance at 280 nm) eluted from thecolumn, the first after about 8 ml of buffer, the second after about 25ml of buffer. Synergizing activity was found only in the second proteinpeak. However, a significant loss (80%) in specific synergizing activitywas observed employing this procedure. Electrophoresis of the fractiondisplaying SAFP activity run using high protein loading (30-50 gprotein/land) was found to contain several higher molecular weightbands.

A second procedure was found to result in improved purification ofzeamatin. Ammonium sulfate fractionation of corn protein extract wasperformed as described above. The dialyzed 30%-55% fraction wassubjected to CM-Sephadex™ chromatography, essentially as describedabove. However, the chromatography was carried out at a slower flow rate(1 ml/min), which resulted in the elution of four distinct peaks (FIG.2). Synergistic anti-Candida activity was confined to peak 3. This peakwas also found to contain growth inhibitory activity against Neurosporacrassa. Anti-Neurospora activity was found only in corn AFPpreparations, not in AFP preparations of wheat and barley.Anti-Trichederma activity was found in all four peaks. Chitinase,glucanase (β1,3- and β1,6-) and β-N-acetylhexosaminidase activities werealso assayed across the four peaks. Chitinase was found in all fourpeaks. A single peak of glucanase activity at fraction 47 and a singlepeak of β-N-acetyl hexosaminidase at fraction 40 were detected.Anti-Neurospora and synergistic anti-Candida activity peaked at fraction44. These antifungal activities did not coincide with any of the enzymeactivities tested.

The fractions associated with synergistic anti-Candida activity (42-48)were combined, concentrated and subjected to further purification.Hydrophobic column chromatography employing phenyl-Sepharose™ (CL-4Bfrom Sigma Chemical Co., St. Louis, Mo.) was used. The sample was loadedonto the column at a low salt concentration employing 0.1M NaCl toreduce the hydrophobic interaction between proteins and the columnmaterials. The elution profile is shown in FIG. 3 (closed circles),compared to the elution profile when higher salt concentrations (1M(NH₄)₂ SO₄) were employed. A large protein fraction passed directlythrough the column (peak 1), followed by another large fraction whosepassage was retarded (peak 2). A third smaller fraction (peak 3) wasthen eluted with 50% ethylene glycol.

The three peak fractions from phenyl-Sepharose™ chromatography wereassayed for enzyme activities and antifungal activities as shown inTable 2. Peak 1 contained most of the chitinase activity, a small amountof glucanase activity, and most of the anti-Trichoderma activity. Noanti-Neurospora or synergistic anti-Candida activity was found inpeak 1. Peak 3 contained most of the glucanase activity and no otherdetectable enzymatic or antifungal activity. Peak 2 contained all of theanti-Neurospora and synergistic anti-Candida activity and a smalleractivity against Trichederma. Peak 2 contained no detectable chitinaseactivity and a very small amount of glucanase.

The purity of the various fractions from the CM-Sephadex™ andnonreducing conditions (FIG. 4). Electrophoresis of the CM-Sephadex™fractions 28, 35, 43, 48 and 52 (FIG. 4A, lanes 1-5 respectively) showeddifferent protein species in the fractions as expected. Combinedfractions 42-48, as well as the phenyl-Sepharose™ fractions peak 1 andpeak 2, were analyzed at low loading (about 5 g protein/lane, FIG. 4A,lanes 6-8) and at 5-fold higher loading (lanes 9-11). In addition, twoseparate phenyl-Sepharose™ chromatography isolates of peak 1 (FIG. 4B,lanes 1 and 2) and peak 2 (FIG. 4B, lanes 3 and 4) and a peak 3 fraction(lane 5) were analyzed. It is apparent that peak 1 contains multipleprotein species present in fractions 42-48, peak 2 contains anapparently homogenous 19 kd protein, zeamatin, and peak 3 contains two30 kd protein species and a small amount of zeamatin. It is estimated atzeamatin isolated in peak 2 of the phenyl-Sepharose™ elution is about95-98% pure.

The protein material isolated from peak 2 of the phenyl-Sepharose™elution was subjected to conventional N-terminal protein sequencing togive the sequence in Table 5.

EXAMPLE 2

In vitro Assays of Antifungal Activity and Synergy

Antifungal activity of protein extracts and fractions was assayed byinhibition of hyphal extension of Trichoderma reesei on agar plates.These assays were performed as described in Roberts and Selitrennikoff,1986 (supra) in 100×15 mm petri plates containing 10 ml of carrot juiceagar medium. Five 0.25 inch diameter sterile blank paper discs wereplaced firmly on the agar, one in the center of a plate and four othersat a distance of 1.2 cm around the central disc. Samples to be testedfor antifungal activity were diluted in buffered saline, 130 mM NaCl/10mM sodium phosphate (pH 7.4), and 25 l portions were added to each ofthe 4 peripheral discs. Control plates were prepared by addition of 25 lof buffered saline to each of the peripheral discs. The concentration ofAFP or SAFP fractions added to discs was measured in g total protein ofthe fraction. Conidia from T. reesi, for example ATCC culture 13631,grown on a suitable agar medium were suspended in 1 ml of bufferedsaline, agitated vigorously to form a slightly turbid suspension and 25l of the suspension was added to the central disc of assay and controlplates. Plates were incubated at 23° C. for approximately 72 h untilmycelial growth from the central disc had enveloped peripheral discs ofthe control plates. The formation of crescents of inhibition aroundsample discs indicated that the sample contained effectiveconcentrations of antifungal agent. Inhibition assays using Neurosporacrassa (for example, 74-OR8-1a, Fungal Genetic Stock Center, HumboldtState College, Arcata, Calif.), Phycomyces blakesleeanus (for example,ATCC No. 8743a) and Alternaria alternaria (for example, ATCC No. 16086)were performed in a similar manner except that growth conditions of thefungi were altered appropriately and Phycomyces was assayed onpotato-dextrose agar medium. All of the grain AFPs acting aloneexhibited antifungal activity toward Trichoderma, Phycomyces, andAlternaria. Wheat and barley AFPs did not inhibit growth of Neurospora.

Inhibition assays of Candida albicans and other yeasts, includingSaccharomyces and Rhodotorula, were performed using a modified discassay in which the organism was suspended in carrot juice agar mediumand potential inhibitors were added to blank paper discs which wereplaced on the agar surface. Candida albicans cultures, for example,swain B-311 (ATCC 32354), were prepared by inoculating 5 ml of sterileliquid medium (1% glucose, 0.5% yeast extract) with a loopful of Candidaand incubating the culture overnight at 37° C. without shaking. Candidasuspension plates ere prepared by adding 1 ml of the overnight cultureof Candida to 100 ml of liquid carrot juice agar at 45°, mixing anddispensing 10 ml of the agar into petri plates. After the Candida agarsuspensions had solidified, five blank paper discs were placed asdescribed above on each plate. Sample and control solutions (30 l) wereadded to discs. Plates were incubated at 37° C. overnight and examinedfor zones of growth inhibition.

In plate assays, fungal inhibition was quantified by measuring thehighest dilution (i.e., lowest total protein concentration) of a samplethat caused a detectable zone of inhibition around an assay disc. Noneof the grain SAFPs alone inhibited growth of Candida, Saccharomyces orRhodotorula in agar plate assays. Inhibition of Candida and Rhodotorulain plate assays was observed only when SAFPs were assayed in thepresence of sub-inhibitory concentrations of antifungal antibiotics,especially nikkomycin.

Antibiotic synergy plate essays were used to assay for SAFP activity.Inhibition by known antifungal antibiotics alone was assayed on platesand quantified, as described above, by adding 30 l of a buffered salinesolution of known concentration of antibiotic to each assay disc. Ininhibition plate assays, the approximate MIC (minimum inhibitoryconcentration) of an antibiotic was defined as the lowest concentrationthat caused a detectable zone of growth inhibition. Synergy ofinhibition of known antifungal antibiotics by protein fractions,extracts or solutions was initially assayed by adding 30 l of a 1:1admixture of antibiotic solution and protein solution to assay discs.Synergy was quantified by comparison of the MIC of the antibiotic aloneto the MIC of the antibiotic/protein composition. Since none of the AFPsor SAFPs alone inhibits growth of C. albicans on plates, under the assayconditions employed any decrease in antibiotic MIC was scored assynergy. A synergy ratio (antibiotic MIC/antibiotic/protein combinationMIC) can be employed for comparisons.

Synergy of inhibition of Candida with nikkomycin in particular, was alsoassessed using a slight modification of the agar plate disc methoddescribed above. A sub-inhibitory level of nikkomycin was added toliquid agar suspensions of C. albicans at 45° C. prior to plating. In astandard assay the final concentration of nikkomycin in the agar wasabout 0.2 g/ml (about 20-fold lower than the concentration of nikkomycinthat alone inhibited the growth of C. albicans). Varying concentrationsof proteins to be tested for synergy were then applied to discs placedon the plates. Synergy was assessed by observation of zones of growthinhibition associated with discs.

Liquid culture assays were also employed to assess antifungal andsynergistic antifungal activity of SAFP, particularly against Candidaalbicans. Liquid culture assays were performed by inoculating theorganism into wells of a 96-well tissue culture plate containing 150 lof fungal growth in each well. Varying concentrations of nikkomycin,SAFP and mixtures of the two agents were introduced into the wells.Plates were incubated in static culture at 37° C. for 24 hours, andwells were then examined visually for culture turbidity. Results of anexperiment assessing zeamatin/nikkomycin activity against C. albicansare shown in Table 3, where growth was scored as +++, ++, + or no growth(-). In this experiment, the initial C. albicans inoculum resulted in anabsorbance reading (650 nm) of about 0.005. The fungal medium employedin this experiment was 2% carrot juice medium.

A fungal growth medium low in salts, devised to promote SAFP activityand yet provide adequate nutrients to permit good fungal growth, wasalso employed in suspension culture assays of antifungal activity. Thismedium contained 0.05% glucose, 10% of the recommended concentration ofMEM amine acids (Flow Laboratories) used to prepare tissue culturemedium, and 10% of the fungal concentration of salts used by Vogel(1964) American Naturalist 98:435-446 to prepare fundal test medium N.T. reesei or N. crassa spores or C. albicans yeast cells were suspendedin this medium at a concentration of approximately 1×10³ organisms/ml.Portions (0.5 ml) of the fungal suspensions were added to wells of atissue culture plate, various additions of protein and/or antibioticwere then added and the plates were incubated at 37° C. for 24 hours.Growth inhibition was defined as no visible growth at the end of thisperiod.

Nikkomycin alone inhibited growth of C. albicans in liquid culture atconcentrations greater than 5 g/ml. Zeamatin alone was also found toinhibit growth of C. albicans in liquid culture. This result wassurprising in light of disc diffusion assays on agar which showed no C.albicans growth inhibition with zeamatin alone. The reason for thesediffering results is not known. In any event, zeamatin displayssynergizing activity with nikkomycin in liquid assays. The MIC ofnikkomycin was reduced from about 17 to about 0.17 g/ml by the additionof 0.3 g/ml of zeamatin. The zeamatin employed in these experiments wasthat purified by phenyl-Sepharose™ chromatography (peak 2, FIG. 3).

A bioautography procedure was developed to assess the presence ofanti-Candida synergy activity in protein bands separated on non-reducingSDS-PAGE. This procedure allowed the specific association of SAFPactivity with a separated protein band on a gel. Protein samples to betested in the bioautography technique were added to non-reducing Laemmlisample buffer (final concentration 15% sucrose, 2.5% SDS, 125 mMTris-HCl, PH 6.7) and boiled for 5 minutes prior to running on a 12%polyacrylamide gel. Gels were then incubated inelution buffer containing1% Triton-X100 for 2 hours at 37° C. to remove SDS. After being washedwell in water, the gels were incubated in 2% carrot juice medium (orother desired growth medium) for 10 minutes. The gels were then placedin 150 mm diameter petri dishes over which an agar solution (1.5% agarin 2% carrot juice medium or other desired fungal growth medium)containing a sub-inhibitory level of nikkomycin (0.2 g/ml and C.albicans (1 ml of an overnight culture to 100 ml of agar solution) waspoured and allowed to solidify. After overnight incubation at 37° C.,the position of SAFP's can be seen as a clear band (no growth) against abackground of Candida growth. Alternatively, the washed protein gels cansimply be placed on a Candida albicans suspension plate containing asub-inhibitory level of nikkomycin and allowed to incubate. Proteinsdiffuse from the gel into the agar to produce a clear zone of growthinhibition that is associated with the protein band. These assays can beemployed with purified protein fractions, partially purified proteinfractions or crude protein extracts.

Protein extracts, fractions and solutions were quantified for totalprotein/ml using the Bradford dye-binding method.

Carrot juice medium was prepared by first autoclaving 20 g of carrotslices in 180 ml water. The resulting carrot juice was then diluted 1:9(v/v) with water, agar (2% w/v) was added and the mixture was againautoclaved. Assays were also performed using a richer nutrient brothagar medium (infra).

Partially purified nikkomycin, which is a combination of nikkomycin Xand Z, approximately 70% pure, was obtained as a gift from Bayer A.-G. Aformulation that includes nikkomycin X and Z is believed to becommercially available in Europe as an agricultural fungicide.Nikkomycin Z and nikkomycin X can be purified from this mixture by knownmethods (Zahner et al. (1981) U.S. Pat. No. 4,287,186). Nikkomycin Z isalso commercially available (Calbiochem, San Diego, Calif., Cat. No.481995). Amphotericin B was obtained from commercial sources (Sigma, St.Louis, Mo.) and papulacandin B, approximately 80% pure, was a gift fromCiba-Geigy Corp. (Basel, Switzerland). Polyoxin B was purified frompolyoxin AL wettable powder as described in Selitrennikoff (1982)Neurospora Newsletter, no. 29, p. 27.

EXAMPLE 3

Chemical and Biological Properties of Zeamatin

The grain SAFPs are all highly basic proteins as evidenced by theirstrong binding to CM-Sephadex™.

Zeamatin partially purified by CM-Sephadex™ (fraction CMS) displayedboth chitinase and β-1,3 glucanase activity in addition to antifungalactivity against T. reesi and N. crassa, and synergistic activity incombination with antifungalantibiotics, especially nikkomycin, againstCandida albicans. Phenyl-Sepharose™-purified zeamatin displayed nochitinase, mannanase or β-N-acetylhexosaminidase activity, and little orno glucanase activity. Synergistic antifungal activity is not associatedwith the presence of chitinase or glucanase activity.

Chitinase, β-1,3 glucanase, β-1,6 glucanase, mannanase andβ-N-acetylhexosaminidase activities were assayed by measuring theincrease of reducing sugar, analogous to the procedure of Dubois et al.(1956) Anal. Chem. 28:350-356. RIP activity of proteins was assessed asdescribed in Coleman and Roberts (1982), supra, by adding dilutions ofthe protein to be assayed to extracts from Ehrlich ascites cells or N.crassa cells, incubating the cell-free reaction mixtures with labelledamino acids, and measuring any protein-induced decrease in incorporationof label into acid insoluble material (i.e., newly synthesized protein).

Trypsin inhibition of proteins was assessed as inhibition of trypsindegradation of the chromogenic trypsin reagent BAPNA(N-α-benzoyl-DL-arginine-p-nitroanilide). Specifically, varying amountsof proteins to be assayed were added to 0.1 ml of trypsin solution (40g/ml in pH 8.0 Tris buffer). The solutions were incubated at roomtemperature for 10 minutes, after which 1.0 ml of BAPNA (0.4 mg/ml) wasadded to each. The reactions were then incubated at 37° C. for anadditional 10 minutes and assayed for production of a yellow color (410nm). Soybean trypsin inhibitor was employed as a positive control (i.e.,no yellow color was produced in the presence of 10 g soybean trypsininhibitor). In the case of zeamatin, addition of 150 g of purifiedzeamatin had no effect on trypsin activity in the assays described.

EXAMPLE 4

Comparison of zeamatin/nikkomycin anti-Candida synergy on differentgrowth media

Synergy assays were performed as described above with Candida, exceptthat assays were also performed on a rich nutrient broth agar medium.Nutrient agar assay plates were prepared as above, substituting acommercial nutrient agar medium for carrot juice agar. Incubation timeswere modified appropriately.

In the growth medium comparison, relative inhibition by nikkomycin Z andzeamatin/nikkomycin Z mixtures was assayed. In zeamatin/nikkomycin Zmixtures, an excess of zeamatin (15 g protein) as fraction CMS was addedto each assay disc and the concentration of nikkomycin Z was varied. TheMIC of nikkomycin Z in the presence and absence of zeamatin wasdetermined as the lowest concentration of the antibiotic that affectsmeasurable growth inhibition of Candida albicans. As shown in Table 4,the MIC of nikkomycin Z against C. albicans grown on nutrient agar wasfound to be about 9 fold higher than the MIC against C. albicans grownon the less-rich carrot juice medium. It is believed that the higher MICon rich medium is due to the presence of inhibitory levels of peptidesin the medium. Zeamatin was found to lower the MIC of Nikkomycin Z onboth rich and poor media. Interestingly, in most cases the nikkomycin ZMIC was lowered about 100 fold in the presence of zeamatin on bothmedia. The minimum mount of partially purified zeamatin (fraction CMS)required to synergize with nikkomycin Z (25 ng/disc or about 0.8 g/ml)was approximately 0.3 g protein per disc (about 10 g/ml) for assayscarried out in both rich and poor media.

EXAMPLE 5

Relative sensitivity of Candida albicans strains to zeamatin/nikkomycincompositions

Several recent clinical isolates of Candida albicans were obtained fromDr. B. Reller, Department of Medicine, University Hospital, Denver,Colo. The sensitivity of the clinical isolates to the synergisticzeamatin/nikkomycin composition was assayed and compared to that of thelaboratory isolate used in the initial assays. Assays were performed asdescribed above, employing nikkomycin Z (Calbiochem) and purifiedzeamatin. Assays were done on carrot juice agar as well as on nutrientbroth agar plates. The results are presented in Table 4. Nikkomycin ZMIC's were determined alone and in the presence of an excess of zeamatin(15 g protein/disc) provided as fraction CMS. There was wide variationin strain sensitivity to both nikkomycin Z alone and zeamatin/nikkomycinZ mixtures. In all cases, the MIC of nikkomycin was lowered in thepresence of zeamatin, and synergy was about as effective on poor mediumas on rich medium.

EXAMPLE 6

Isolation and Purification of Sormatin from Sorghum

Sormatin was isolated and purified from sorghum following a proceduresimilar to that used in the isolation and purification of zeamatin fromcorn. Specifically, Pioneer Hi-Bred #8333 sorghum was ground freely in acoffee grinder. Sorghum meal was extracted for 1 h at 4° C. with abuffer containing 50 mM NaCl, 25 mM NaPO₄, 5 Mm EDTA (pH 7.4). Theresulting suspension was centrifuged (10,000×g, 20 min, 4° C.) and thesupernatant protein extract was saved.

Ammonium sulfate was slowly added with stirring to the sorghum proteinextract, to obtain a 35% saturated solution which was left overnight at40° C. The solution was then centrifuged (10,000×g, 30 min, 4° C.), thepellet discarded and the supernatant was made 65% saturated withammonium sulfate. The solution was left 20 min at 4° C., centrifuged(10,000×g, 30 min, 4° C.) and the supernatant was discarded. Theremaining protein pellet was resuspended in buffer containing 10 mMNaCl, 5 mM NaPO₄, 1 mM EDTA (PH 7.4) and dialyzed for about 18 h at 4°C. with four changes of the same resuspension buffer. The dialyzedsolution was clarified by centrifugation (10,000×g, 20 min, 4° C.). ACM-Sephadex™ column (about 60 ml of packed gel volume) was equilibratedwith resuspension buffer. The dialyzed solution was loaded onto thecolumn; the column was then washed with buffer until the absorbance ofthe effluent at 280 nm had fallen to about 0.2. A linear NaCl gradient(resuspension buffer (pH 7.4) plus NaCl) from 10 nM NaCl gradient(resuspension buffer (pH 7.4) plus NaCl) from 10 mM NaCl to 400 mM NaClwas applied to the column as described in Example 1 employing the slowerflow rate of about 1 ml/min. FIG. 5 shows the elution profile from theCM-Sephadex™ column purification of sormatin comprising four peaks. Thefractions comprising peak III were concentrated using an Amicon YM-10filter and the concentrate was loaded on a phenyl-Sepharose™ (CL-4B)column. Phenyl-Sepharose™ chromatography was carried out as described inExample 1 for zeamatin. The sample was loaded onto the column at a lowsalt concentration employing 0.1M NaCl. The elution profile is shown inFIG. 6. Only peak B contained synergistic anti-Candida activity. FIG. 7shows SDS-polyacrylamide gel electrophoresis under reducing conditionsof crude sorghum extract and various protein fractions. Lane 9 is thephenyl-Sepharose™ peak B fraction containing the approximately 25 kdsorghum protein having synergistic antifungal activity. Sormatin isbelieved to be about 70-80% pure after phenyl-Sepharose™ chromatography.

The protein material isolated from peak B, sormatin, was subjected toconventional N-terminal protein sequencing techniques to obtain thesequence given in Table 5.

Sormatin, either in purified or partially purified form, has synergisticantifungal activity similar to that of zeamatin.

EXAMPLE 7

Isolation and Partial Purification of SAFP from Oats

Avematin from oat meal was isolated and purified to the CM-Sephadex™step as described above for zeamatin employing the same bufferextraction procedure (buffer pH 7.0) and ammonium sulfate fractionation.CM-Sephadex™ chromatography was performed using a linear salt gradient(10 mM NaCl-400 mM NaCl) employing 1 ml/min flow rate to give theCM-Sephadex™ elution profile given in FIG. 9. Peak I of FIG. 9 was foundto contain synergistic antifungal activity, particularly anti-Candidaactivity. FIG. 10 shows the results of SDS polyacrylamide gelelectrophoresis under reducing conditions of peak I of FIG. 9. Avematinis believed to be about 50% pure when purified by this CM-Sephadex™procedure.

Avematin in partially purified form has synergistic antifungal activityand antifungal activity similar to zeamatin.

EXAMPLE 8

SAFP Activity In Non-Grain Plant Extracts

As described above, SAFP activity has been detected in crude andpartially purified extracts of a variety of grains. Using in vitrosynergy assays as described in Example 2, extracts of other plants canbe assessed for the presence of SAFP activity. For example, SAFPactivity has been detected in extracts of soybeans. Soybeans were groundand extracted wish buffer (50 mM NaCl, 20 mM NaPO₄ buffer, 5 mM EDTA, pH7.4) and the extract clarified by centrifugation (10,000×g, 20 m, 4°C.). The crude extract was found to synergize with nikkomycin against C.albicans in in vitro plate assays. Thus, SAFP activity is not limited tograins and is likely to be found in a variety of other plant seeds andother plant materials.

EXAMPLE 9

Protein Sequence Determination of Zeamatin

Example 1 describes the purification of zeamatin and the determinationof the aminoterminal sequence of the zeamatin protein (see table 5). Inaddition, purified zeamatin and chymotrypsin digestion products weresubjected to amino acid sequence determination using techniques known inthe art. The protein sequence was found to be identical to the sequencepublished by Richardson et al. and from the Richardson sequence theentire 206 amino acid sequence can be predicted. This is shown in table6. Underlined sequences are those determined from chymotryptic digestionproducts. The mature protein starts on an alanine as a 21 amino acidsignal sequence is removed from the pre-protein.

                                      TABLE 6                                     __________________________________________________________________________    Amino Acid Sequences of Zeamatin (SEQ ID NO:3)                                 1  AVFTVVNQCPFTVWAASVPVGGGRQLNRGESWRITAPAGTTAARIWARTG                         51 CQFDASGRGSCRTGDCGGVVQCTGYGRAPNTLAEYALKQFNNLDFFDISI                        101 LDGFNVPYSFLPDGGSGCSRGPRCAVDVNARCPAELRQDGVCNNACPVFK                        151 KDEYCCVGSAANNCHPTNYSRYFKGQCPDAYSYPKDDATSTFTCPAGTNY                        201 KVVFCP                                                                    __________________________________________________________________________

EXAMPLE 10

Cloning of a Zeamatin cDNA

Based on the complete amino acid sequence of zeamatin (see example 9)the following degenerate oligonucleotides were designed:

SP86 ATGTGAATTCGGICAIAAIACYTTRTARTT (SEQ ID NO:4)

SP87 ATGTGAATTCAICAITAYTCITCYTTYTTIAA (SEQ ID NO:5)

SP88 GTACGGATCCAAYCAITGYCCITTYACIGTITGG (SEQ ID NO:6)

From the amino acid sequence SP86 and SP88 would predictably amplify aDNA fragment of 600 bp, and SP87 and SP88 a fragment of 450 bp from thezeamatin cDNA. Oligonucleotides SP86 and SP87 included an EcoRI site attheir 5' terminus and oligonucleotide SP88 included a BamHI site at its5' terminus. These restriction sites are for the purposes offacilitating the cloning of the PCR fragments generated. Note thatY=pyrimidine, R=purine, and I=inosine.

First strand cDNA was synthesized from polyA+RNA from 6 day-oldlight-grown maize shoots and used as a template in the polymerase chainreaction using 40 cycles of 94° C./1 min., 50° C./1.5 min. Bothogilonucleotides generated fragments of the predicted size, which weregel-purified and reamplified. The fragments were subsequently clonedinto pBluescript and sequence determination of the resultant plasmidsconfirmed that the correct zeamatin sequence had been amplified.

Probes generated from the zeamatin cloned sequences were radio-labelledand used to screen a cDNA library (in λZiplox) from 7-day old maize B73.Three separate full-length cDNA clones were isolated and, following invivo excision, the DNA sequence of each was determined. All three cloneswere found to be identical in sequence. The full-length zeamatin cDNA ispresented in Table 7.

                                      TABLE 7                                     __________________________________________________________________________    cDNA Sequence of Zeamatin (SEQ ID NO:7)                                       __________________________________________________________________________     1 TTCAAATACT                                                                            TGCATAAGAT                                                                            GGCAGGTTCC                                                                            GTGGCAATCG                                                                            TGGGCATCTT                                  51                                                                              CGTCGCCCTC                                                                            CTCGCCGTGG                                                                            CCGGCGAGGC                                                                            GGCTGTGTTC                                                                            ACGGTGGTGA                                 101                                                                              ACCAGTGCCC                                                                            GTTCACCGTG                                                                            TGGGCCGCGT                                                                            CCGTGCCGGT                                                                            GGGCGGCGGG                                 151                                                                              CGGCAGCTGA                                                                            ACCGCGGCGA                                                                            GAGCTGGCGG                                                                            ATCACGGCCC                                                                            CCGCGGGCAC                                 201                                                                              GACGGCCGCG                                                                            CGCATCTGGG                                                                            CGCGCACGGG                                                                            GTGCAAGTTC                                                                            GACGCGAGCG                                 251                                                                              GGCGCGGGAG                                                                            CTGCCGCACG                                                                            GGGGACTGCG                                                                            GCGGCGTGCT                                                                            GCAGTGCACT                                 301                                                                              GGGTACGGGC                                                                            GCGCGCCCAA                                                                            CACGCTGGCG                                                                            GAGTACGCTC                                                                            TGAAGCAGTT                                 351                                                                              CACAACCTCG                                                                            ACTTCTTCGA                                                                            CATCTCCCTC                                                                            ATCGACGGCT                                                                            TCAACGTGCC                                 401                                                                              CATGAGCTTC                                                                            CTCCCCGACG                                                                            GCGGGTCCGG                                                                            GTGCAACCGC                                                                            GGCCCGCGCT                                 451                                                                              GCGCCGTGGA                                                                            CGTGAACGCG                                                                            CGCTGCCCTG                                                                            CCGAGCTGCG                                                                            GCAGGACGGC                                 501                                                                              GTGTGCAACA                                                                            ACGCGTGCCC                                                                            CGTGTTCAAG                                                                            AAGGACGAGT                                                                            ACTGCTGCGT                                 551                                                                              CGGCTCGGCG                                                                            GCCAACGACT                                                                            GCCACCCGAC                                                                            CAACTACTCC                                                                            AGGTACTTCA                                 601                                                                              AGGGGCAGTG                                                                            CCCCGACGCG                                                                            TACAGCTACC                                                                            CCAAGGATGA                                                                            CGCCACCAGC                                 651                                                                              ACCTTCACCT                                                                            GCCCCGCCGG                                                                            AACCAACTAC                                                                            AAGGTCGTCT                                                                            TCTGCCCGTG                                 701                                                                              AGGCCGCTGA                                                                            ACTAGCATCA                                                                            GCGTGCGCGC                                                                            GTCGACCAAG                                                                            AACAAGAAAT                                 751                                                                              AAACGACCGA                                                                            GGCTGTGTAT                                                                            GTTGCCCGCC                                                                            GTGTGCTCAA                                                                            CTGGAGCAAA                                 801                                                                              AATAAAGCCA                                                                            ACAAAACAAG                                                                            CACGTGTGAT                                                                            CTCTATGTCA                                                                            CCAACTCTAT                                 851                                                                              CACCTTAATT                                                                            AATGGGGCAT                                                                            TATGAAA AAAAAAA AAAA                                       __________________________________________________________________________

The ATG codon for the first methionine and the first codon of the maturepeptide are underlined.

EXAMPLE 11

Expression of the Zeamatin cDNA in Transgenic Plants

The cDNA for zeamatin was expressed behind the constitutive 35S promoterin transgenic plants. The 913 bp cDNA was transferred as an EcoRI-NotIfragment from pBluescript to the vector pCGN1761/ENX which carries thedouble 35S CaMV promoter and the tml transcriptional terminator on apUC-derived plasmid. Colonies carrying the cDNA in sense were recoveredand named pZMT-A. The expression cassette of pZMT-A was subsequentlyexcised as an XbaI fragment and cloned into pCIB200 and coloniesoriented in such a way that the 35S promoter was located adjacent to theselectable marker were used in plant transformations usingAgrobacterium. For direct gene transfer, the pZMT-A expression cassetteis transferred as a HindIII-BamHI fragment to the vector pCIB3064.Transformation to transgenic plants is undertaken using techniques wellknown in the art. For transformation of dicotyledonous species usingbinary Agrobacterium vectors such as pCIB200 see Alexander et al. (PNASin press), and for transformation of monocotyledonous species usingdirect gene transfer vectors such as pCIB3064 see Koziel et al. (1993;Biotechnology 11: 194-200). Transgenic plants are screened forhigh-level expression of the zeamatin cDNA using antibodies whichrecognize the zeamatin protein or by Northern analysis. Plants whichexpress high levels of the zeamatin protein are found to have enhancedresistance to plant pathogens.

Other promoters are suitable for the expression of zeamatin intransgenic plants. These include (but are not restricted to)constitutive promoters (such as those from the ubiquitin and actingenes), cell and tissue-specific promoters (examples), and promoterswhich are expressed upon wound induction, infection or upon initiationof the plant response to infection (such as the tobacco PR1 promoter).

Other synergistic antifungal protein cDNAs can be expressed in trangenicplants in a manner analogous to that described for zeamatin in example11.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..30                                                           (D) OTHER INFORMATION: /note= "N-terminal amino acid                          sequence of Zeamatin"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaValPheThrValValAsnGlnCysProPheThrValTrpAlaAla                              151015                                                                        SerValProValGlyGlyGlyArgGlnLeuAsnArgGlyGlu                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note= "N-terminal amino acid                          sequence of Sormatin"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaValPheThrValValAsnArgCysProTyrThrValTrpAlaAla                              151015                                                                        SerValProValGlyGly                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 206 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..206                                                          (D) OTHER INFORMATION: /note= "Amino acid sequence for                        Zeamatin protein. Mature protein begins at                                    residue 21."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaValPheThrValValAsnGlnCysProPheThrValTrpAlaAla                              151015                                                                        SerValProValGlyGlyGlyArgGlnLeuAsnArgGlyGluSerTrp                              202530                                                                        ArgIleThrAlaProAlaGlyThrThrAlaAlaArgIleTrpAlaArg                              354045                                                                        ThrGlyCysGlnPheAspAlaSerGlyArgGlySerCysArgThrGly                              505560                                                                        AspCysGlyGlyValValGlnCysThrGlyTyrGlyArgAlaProAsn                              65707580                                                                      ThrLeuAlaGluTyrAlaLeuLysGlnPheAsnAsnLeuAspPhePhe                              859095                                                                        AspIleSerIleLeuAspGlyPheAsnValProTyrSerPheLeuPro                              100105110                                                                     AspGlyGlySerGlyCysSerArgGlyProArgCysAlaValAspVal                              115120125                                                                     AsnAlaArgCysProAlaGluLeuArgGlnAspGlyValCysAsnAsn                              130135140                                                                     AlaCysProValPheLysLysAspGluTyrCysCysValGlySerAla                              145150155160                                                                  AlaAsnAsnCysHisProThrAsnTyrSerArgTyrPheLysGlyGln                              165170175                                                                     CysProAspAlaTyrSerTyrProLysAspAspAlaThrSerThrPhe                              180185190                                                                     ThrCysProAlaGlyThrAsnTyrLysValValPheCysPro                                    195200205                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Degenerate nucleotide SP86 based on                          amino acid sequence of zeamatin                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 16                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 19                                                              (D) OTHER INFORMATION: /mod_base=i                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGTGAATTCGGNCANAANACYTTRTARTT30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Degenerate nucleotide SP87 based on                          amino acid sequence of zeamatin                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 30                                                              (D) OTHER INFORMATION: /mod_base=i                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGTGAATTCANCANTAYTCNTCYTTYTTNAA32                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Degenerate nucleotide SP88 based on                          amino acid sequence of zeamatin                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 16                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 22                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 28                                                              (D) OTHER INFORMATION: /mod_base=i                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified_base                                                   (B) LOCATION: 31                                                              (D) OTHER INFORMATION: /mod_base=i                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GTACGGATCCAAYCANTGYCCNTTYACNGTNTGG34                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 894 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 19..21                                                          (D) OTHER INFORMATION: /note= "Start codon for coding                         sequence of Zeamatin"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 82..84                                                          (D) OTHER INFORMATION: /note= "First codon of the mature                      peptide for Zeamatin"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TTCAAATACTTGCATAAGATGGCAGGTTCCGTGGCAATCGTGGGCATCTTCGTCGCCCTC60                CTCGCCGTGGCCGGCGAGGCGGCTGTGTTCACGGTGGTGAACCAGTGCCCGTTCACCGTG120               TGGGCCGCGTCCGTGCCGGTGGGCGGCGGGCGGCAGCTGAACCGCGGCGAGAGCTGGCGG180               ATCACGGCCCCCGCGGGCACGACGGCCGCGCGCATCTGGGCGCGCACGGGGTGCAAGTTC240               GACGCGAGCGGGCGCGGGAGCTGCCGCACGGGGGACTGCGGCGGCGTGCTGCAGTGCACT300               GGGTACGGGCGCGCGCCCAACACGCTGGCGGAGTACGCTCTGAAGCAGTTCACAACCTCG360               ACTTCTTCGACATCTCCCTCATCGACGGCTTCAACGTGCCCATGAGCTTCCTCCCCGACG420               GCGGGTCCGGGTGCAACCGCGGCCCGCGCTGCGCCGTGGACGTGAACGCGCGCTGCCCTG480               CCGAGCTGCGGCAGGACGGCGTGTGCAACAACGCGTGCCCCGTGTTCAAGAAGGACGAGT540               ACTGCTGCGTCGGCTCGGCGGCCAACGACTGCCACCCGACCAACTACTCCAGGTACTTCA600               AGGGGCAGTGCCCCGACGCGTACAGCTACCCCAAGGATGACGCCACCAGCACCTTCACCT660               GCCCCGCCGGAACCAACTACAAGGTCGTCTTCTGCCCGTGAGGCCGCTGAACTAGCATCA720               GCGTGCGCGCGTCGACCAAGAACAAGAAATAAACGACCGAGGCTGTGTATGTTGCCCGCC780               GTGTGCTCAACTGGAGCAAAAATAAAGCCAACAAAACAAGCACGTGTGATCTCTATGTCA840               CCAACTCTATCACCTTAATTAATGGGGCATTATGAAAAAAAAAAAAAAAAAAAA894                     __________________________________________________________________________

We claim:
 1. A method of inhibiting the growth of a fungus whichcomprises applying to said fungus or a habitat of said fungus zeamatinin substantially pure form in an antifungally effective amount or anamount sufficient to enhance the antifungal activity of an antifungalantibiotic selected from the group of antifungal antibiotics whichcomprises nikkomycins, polyoxins and amphotericins, wherein saidzeamatin is a protein isolatable from corn, having a molecular weight ofabout 22 kD under reducing conditions upon SDS-PAGE, and having theN-terminal amino acid sequence A V F V V N Q C P F T V W A A S V P V G GG R Q L R G E.
 2. The method of claim 1 wherein said fungus is a strainselected from the group consisting of strains of Candida, TricholermaNeurospora, Rhizoctonia, Fusarium and Chaetomium.
 3. The method of claim1 wherein said fungus is a plant pathogen.
 4. The method of claim 1wherein said zeamatin protein is obtainable from corn steepwater or aprotein concentrate thereof.
 5. A method of inhibiting the growth of afungus which comprises applying to said fungus or a habitat of saidfungus sormatin in substantially pure form in an antifungally effectiveamount or an amount sufficient to enhance the antifungal activity of anantifungal antibiotic selected from the group of antifungal antibioticswhich comprises nikkomycins, polyoxins and amphotericins, wherein saidsormatin is a protein isolatable from sorghum, having a molecular weightof about 25 kD under reducing conditions upon SDS-PAGE, and having theN-terminal amino acid sequence A V F T V V N R C P Y T V W A A S V P V GG.
 6. The method of claim 5 wherein said fungus is a strain selectedfrom the group consisting of strains of Candida, Trichoderma Neurospora,Rhizoctonia, Fusarium and Chaetomium.
 7. The method of claim 5 whereinsaid fungus is a plant pathogen.
 8. The method of claim 5 wherein saidsormatin protein is obtainable from a partially purified protein extractof sorghum.
 9. A method of inhibiting the growth of a fungus whichcomprises applying to said fungus or a habitat of said fungus avematinin partially purified form in an antifungally effective amount or anamount sufficient to enhance the antifungal activity of an antifungalantibiotic selected from the group of antifungal antibiotics whichcomprises nikkomycins, polyoxins and amphotericins, wherein saidavematin is a protein isolatable from oat and having a molecular weightof about 22 kD under reducing conditions upon SDS-PAGE.
 10. The methodof claim 9 wherein said fungus is a strain selected from the groupconsisting of strains of Candida, Trichoderma Neurospora, Rhizoctonia,Fusarium and Chaetomium.
 11. The method of claim 9 wherein said fungusis a plant pathogen.
 12. A method of inhibiting the growth of a funguswhich comprises applying to said fungus or a habitat of said fungus anantifungal composition which comprises an antifungal antibiotic and anantifungal protein, said antifungal antibiotic and said antifungalprotein being present in concentrations sufficient to inhibit the growthof said fungus, said antifungal protein being a protein selected from(i)zeamatin isolatable from corn, having a molecular weight of about 22 kDunder reducing conditions upon SDS-PAGE, and having the N-terminal aminoacid sequence A V F T V V N Q C P F T V W A A S V P V G G G R Q L N R GE; (ii) sormatin isolatable from sorghum, having a molecular weight ofabout 25 kD under reducing conditions upon SDS-PAGE, and having theN-terminal amino acid sequence A V F T V V N R C P Y T V W A A S V P V GG; and (iii) avematin isolatable from oat and having a molecular weightof about 22 kD under reducing conditions upon SDS-PAGE;and saidantifungal antibiotic being selected from the group of antifungalantibiotics which comprise nikkomycins, polyoxins and amphotercins. 13.The method of claim 12 in which said fungus is selected from the groupof fundi consisting of Candida and Rhodotorula.
 14. The method of claim13 in which said fungus is a strain of Candida.
 15. The method of claim14 in which said fungus is a strain of Candida albicans.
 16. The methodof claim 12 wherein said antifungal antibiotic of said composition is anikkomycin.
 17. The method of claim 12 wherein said nikkomycin ispresent at a concentration greater than or equal to about 0.03 times theMIC of said nikkomycin alone.
 18. The method of claim 12 wherein saidnikkomycin is present at a concentration greater than or equal to about0.01 times the MIC of said nikkomycin alone.
 19. A method according toclaim 12 wherein the fungus is a strain of Candida albicans, theantifungal antibiotic is a nikkomycin that has anti-Candida activity,and the antifungal protein is zeamatin.
 20. The method of claim 19wherein said nikkomycin is nikkomycin X.
 21. The method of claim 19wherein said nikkomycin is nikkomycin Z.
 22. The method of claim 19wherein said nikkomycin is present at a concentration greater than orequal to about 0.03 times the MIC of said nikkomycin alone.
 23. Themethod of claim 19 wherein said nikkomycin is present at a concentrationgreater than or equal to about 0.01 times the MIC of said nikkomycinalone.
 24. The method of claim 19 wherein the zeamatin is present inamounts sufficient to lower the MIC of nikkomycin by about 33-100 foldrelative to the MIC of nikkomycin alone for said strain of Candidaalbicans.
 25. A method according to claim 12 wherein the fungus is astrain of Candida albicans, the antifungal antibiotic is a nikkomycinhaving anti-Candida activity, and the antifungal protein is sormatin.26. A method according to claim 12 wherein the fungus is a strain ofCandida albicans, the antifungal antibiotic is a nikkomycin that hasanti-Candida activity, and the antifungal protein is avematin.